Serine protease inhibitor for treating coronavirus infection

ABSTRACT

The disclosure is directed to WX-671, as (L)- or (D)-enantiomers, and as E- or (Z)-isomers or (E/Z)-mixtures, and as free bases or as salts thereof, in preparing medicines for treating coronavirus infection or preventing diseases caused by coronavirus infection, and a medicine for preventing coronavirus infection or preventing diseases caused by coronavirus infection.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/987,429, filed Mar. 10, 2020; U.S. Provisional Application No.63/003,601, filed Apr. 1, 2020; U.S. Provisional Application No.63/034,817 filed Jun. 4, 2020; U.S. Provisional Application No.63/074,799, filed Sep. 4, 2020; and U.S. Provisional 63/125,427 filedDec. 15, 2020. These applications are incorporated by reference in theirentireties for all purposes.

BACKGROUND

Coronaviruses are lipid enveloped positive-stranded RNA viruses (+ssRNA) that replicate in the cell cytoplasm. Prior to 2002, coronaviruseswere not considered to be significant human pathogens. Other humancoronaviruses such as HCoV-229E and HCoV-OC43 resulted in only mildrespiratory infections in healthy adults. In 2002, however, severe acuterespiratory syndrome coronavirus (SARS-CoV) emerged in GuangdongProvince, China. While SARS-CoV predominantly impacted Southeast Asia,with significant outbreaks throughout China, Hong Kong, Taiwan,Singapore, and Vietnam, the virus was carried outside the region.

In 2012, Middle East respiratory syndrome coronavirus (MERS-CoV), wasdetected in a patient with severe respiratory disease in Saudi Arabia.The clinical features of MERS-CoV infection in humans range fromasymptomatic to very severe pneumonia with the potential development ofacute respiratory distress syndrome, septic shock, and multiorganfailure resulting in death. Since the first case of MERS-CoV infectionwas reported and the virus was isolated, significant progress has beenmade toward understanding the epidemiology, ecology, and biology of thevirus. Several assays for the detection of acute infection with MERS-CoVby real-time reverse transcription (RT)-PCR have been developed and arein widespread use.

In 2019, a novel coronavirus (nCoV) emerged in the world and is nowknown to cause coronavirus disease 2019 (COVID-19). COVID-19 is aninfectious disease caused by severe acute respiratory syndromecoronavirus 2 (SARS coronavirus-2 or SARS-CoV-2), a virusphylogenetically closely related to SARS virus. The World HealthOrganization (WHO) has declared the 2019-2020 coronavirus outbreak to bea Public Health Emergency of International Concern (PHEIC). For mostpatients, COVID-19 begins and ends in their lungs, because coronavirusesprimarily cause respiratory diseases.

SUMMARY

The present invention relates generally to the fields of virology,infectious disease and medicine. The invention provides a new use ofWX-671 as (L)- or (D)-enantiomers, and as E- or (Z)-isomers or(E/Z)-mixtures, and as free bases or as salts thereof, in thepreparation of medicines for treating coronavirus infection in humans.

According to aspects illustrated herein, there is disclosed a method forthe treatment of the 2019 coronavirus disease (COVID-19) caused by theSARS-CoV-2 virus in a human in need thereof that includes administeringan effective amount of WX-671,

as (L)- or (D)-enantiomers, and as E- or (Z)-isomers or (E/Z)-mixtures,and as free bases or as salts thereof. In an embodiment, WX-671 existsas a hydrogen sulfate salt. In an embodiment, WX-671 is combined with apharmaceutically-acceptable carrier material. In an embodiment, theWX-671 and optionally the pharmaceutically-acceptable carrier material,are in a unit dosage form suitable for oral administration. In anembodiment, the dosage form is a solid dosage form. In an embodiment,the solid dosage form is a capsule. In an embodiment, the SARS-CoV-2virus is wild-type. In an embodiment, the SARS-CoV-2 virus is anaturally occurring coronavirus variant. In an embodiment, 200 mg ofWX-671 is administered in a single capsule to a human in need thereof,once a day for at least 10 days, for a total daily dose of 200 mg. In anembodiment, 400 mg of WX-671 is administered in two capsules to a humanin need thereof, once a day for at least 10 days, for a total daily doseof 400 mg. In an embodiment, about 231 mg of WX-671.1 (upamostat) isadministered in a single capsule to a human in need thereof, once a dayfor at least 10 days, for a total daily dose equivalent to 200 mg of thefree form. In an embodiment, about 463 mg of WX-671.1 (upamostat) isadministered as two capsules to a human in need thereof, once a day forat least 10 days, for a total daily dose equivalent to 400 mg of thefree form. In an embodiment, administration of the effective amount ofWX-671 results in a decrease of viral load by at least 10%.

According to aspects illustrated herein, there is disclosed a method oftreatment comprising administering an effective amount of WX-671,

as (L)- or (D)-enantiomers, and as E- or (Z)-isomers or (E/Z)-mixtures,and as free bases or as salts thereof, to a human having 2019coronavirus disease (COVID-19) caused by the SARS-CoV-2 virus. In anembodiment, WX-671 exists as a hydrogen sulfate salt. In an embodiment,WX-671 is combined with a pharmaceutically-acceptable carrier material.In an embodiment, WX-671 optionally with a pharmaceutically-acceptablecarrier material, are in a unit dosage form suitable for oraladministration. In an embodiment, the dosage form is a solid dosageform. In an embodiment, the solid dosage for is a capsule. In anembodiment, the SARS-CoV-2 virus is wild-type. In an embodiment, theSARS-CoV-2 virus is a naturally occurring coronavirus variant. In anembodiment, WX-671.1 (upamostat) is administered as a single capsulecomprising 200 mg of the free base, and wherein a single capsule isadministered to a human in need thereof once a day for at least 10 days,for a total daily dose of 200 mg. In an embodiment, WX-671.1 (upamostat)is administered as two capsules, each capsule comprising 200 mg, andwherein two capsules are administered to a human in need thereof once aday for at least 10 days, for a total daily dose of 400 mg.

According to aspects illustrated herein, there is disclosed WX-671,

as (L)- or (D)-enantiomers, and as E- or (Z)-isomers or (E/Z)-mixtures,and as free bases or as salts thereof, for use in treating coronavirusinfection.

According to aspects illustrated herein, there is disclosed WX-671,

as (L)- or (D)-enantiomers, and as E- or (Z)-isomers or (E/Z)-mixtures,and as free bases or as salts thereof, for use in treating the 2019coronavirus disease (COVID-19) caused by the SARS-CoV-2 virus.

According to aspects illustrated herein, there is disclosed(N-α-(2,4,6-triisopropylphenylsulfonyl)-3-hydroxyamidino-phenylalanine-4-ethoxycarbonylpiperazide)as (L)- or (D)-enantiomers, and as E- or (Z)-isomers or (E/Z)-mixtures,and as free bases or as salts thereof, for use in treating coronavirusinfection.

According to aspects illustrated herein, there is disclosed(N-α-(2,4,6-triisopropylphenylsulfonyl)-3-hydroxyamidino-phenylalanine-4-ethoxycarbonylpiperazide)as (L)- or (D)-enantiomers, and as E- or (Z)-isomers or (E/Z)-mixtures,and as free bases or as salts thereof, for use in treating the 2019coronavirus disease (COVID-19) caused by the SARS-CoV-2 virus.

According to aspects illustrated herein, there is disclosed use ofWX-671,

as (L)- or (D)-enantiomers, and as E- or (Z)-isomers or (E/Z)-mixtures,and as free bases or as salts thereof, for the manufacture of amedicament for treatment of coronavirus infection.

According to aspects illustrated herein, there is disclosed use ofWX-671,

as (L)- or (D)-enantiomers, and as E- or (Z)-isomers or (E/Z)-mixtures,and as free bases or as salts thereof, for the manufacture of amedicament for treatment of the 2019 coronavirus disease (COVID-19)caused by the SARS-CoV-2 virus.

According to aspects illustrated herein, there is disclosed use of(N-α-(2,4,6-triisopropylphenylsulfonyl)-3-hydroxyamidino-phenylalanine-4-ethoxycarbonylpiperazide)as (L)- or (D)-enantiomers, and as E- or (Z)-isomers or (E/Z)-mixtures,and as free bases or as salts thereof, for the manufacture of amedicament for treatment of coronavirus infection.

According to aspects illustrated herein, there is disclosed use of(N-α-(2,4,6-triisopropylphenylsulfonyl)-3-hydroxyamidino-phenylalanine-4-ethoxycarbonylpiperazide)as (L)- or (D)-enantiomers, and as E- or (Z)-isomers or (E/Z)-mixtures,and as free bases or as salts thereof, for the manufacture of amedicament for treatment of the 2019 coronavirus disease (COVID-19)caused by the SARS-CoV-2 virus.

According to aspects illustrated herein, there is disclosed apharmaceutical composition for the treatment of coronavirus infection,comprising WX-671,

as (L)- or (D)-enantiomers, and as E- or (Z)-isomers or (E/Z)-mixtures,and as free bases or as salts thereof.

According to aspects illustrated herein, there is disclosed apharmaceutical composition for the treatment of the 2019 coronavirusdisease (COVID-19) caused by the SARS-CoV-2 virus, comprising WX-671,

as (L)- or (D)-enantiomers, and as E- or (Z)-isomers or (E/Z)-mixtures,and as free bases or as salts thereof.

According to aspects illustrated herein, there is disclosed apharmaceutical composition for the treatment of coronavirus infection,comprising(N-α-(2,4,6-triisopropylphenylsulfonyl)-3-hydroxyamidino-phenylalanine-4-ethoxycarbonylpiperazide)as (L)- or (D)-enantiomers, and as E- or (Z)-isomers or (E/Z)-mixtures,and as free bases or as salts thereof.

According to aspects illustrated herein, there is disclosed apharmaceutical composition for the treatment of the 2019 coronavirusdisease (COVID-19) caused by the SARS-CoV-2 virus,(N-α-(2,4,6-triisopropylphenylsulfonyl)-3-hydroxyamidino-phenylalanine-4-ethoxycarbonylpiperazide)as (L)- or (D)-enantiomers, and as E- or (Z)-isomers or (E/Z)-mixtures,and as free bases or as salts thereof.

According to aspects illustrated herein, there is disclosed ananti-coronavirus infection agent comprising WX-671,

as (L)- or (D)-enantiomers, and as E- or (Z)-isomers or (E/Z)-mixtures,and as free bases or as salts thereof.

According to aspects illustrated herein, there is disclosed ananti-coronavirus infection agent comprising(N-α-(2,4,6-triisopropylphenylsulfonyl)-3-hydroxyamidino-phenylalanine-4-ethoxycarbonylpiperazide)as (L)- or (D)-enantiomers, and as E- or (Z)-isomers or (E/Z)-mixtures,and as free bases or as salts thereof.

According to aspects illustrated herein, there is disclosed a method forthe treatment of human coronavirus infection, comprising administeringto a subject in need thereof a therapeutically effective amount of acompound selected from one ofN-α(2,4,6-triisopropylphenylsulfonyl)-3-amidino-phenylalanine-4-ethoxy-carbonylpiperazide-hydrochlorideor its prodrugN-α-(2,4,6-triisopropylphenylsulfonyl)-3-hydroxyamidino-phenylalanine-4-ethoxycarbonylpiperazide,wherein the selected compound can be present as (L)- or (D)-enantiomers,and as E- or (Z)-isomers or (E/Z)-mixtures, and as free bases or assalts thereof. In an embodiment, the method further comprisesdiagnostically confirming that the subject is infected with a humancoronavirus prior to administering the compound. In an embodiment, thecoronavirus infection is severe acute respiratory syndrome coronavirus 2(SARS-CoV-2). In an embodiment, the compound isN-α-(2,4,6-triisopropylphenylsulfonyl)-3-hydroxyamidino-phenylalanine-4-ethoxycarbonylpiperazideand in an orally administrable form. In an embodiment, the compound isN-α(2,4,6-triisopropylphenylsulfonyl)-3-amidino-phenylalanine-4-ethoxy-carbonylpiperazide-hydrochlorideand in an injectable form to be delivered intravenously orintramuscularly. In an embodiment, the compound isN-α-(2,4,6-triisopropylphenylsulfonyl)-3-hydroxyamidino-phenylalanine-4-ethoxycarbonylpiperazidepresent as a sulfate or hydrogen sulfate salt. In an embodiment, thecompound isN-α-(2,4,6-triisopropylphenylsulfonyl)-3-hydroxyamidino-phenylalanine-4-ethoxycarbonylpiperazidepresent in the L-stereoisomer conformation. In an embodiment, thecompound isN-α-(2,4,6-triisopropylphenylsulfonyl)-3-hydroxyamidino-(L)-phenylalanine-4-ethoxycarbonylpiperaziniumhydrogen sulfate. In an embodiment, the compound isN-α-(2,4,6-triisopropylphenylsulfonyl)-3-hydroxyamidino-phenylalanine-4-ethoxycarbonylpiperazideand is to be administered in a dose of 200 mg per day. In an embodiment,the compound isN-α-(2,4,6-triisopropylphenylsulfonyl)-3-hydroxyamidino-phenylalanine-4-ethoxycarbonylpiperazideand is to be administered in a dose of 400 mg per day.

According to aspect illustrated herein, there is disclosed a method oftreating COVID-19 (SARS-CoV-2) coronavirus infection, the methodcomprising administering to a subject in need thereof for at least 14days one or more therapeutically effective doses of a compound selectedfrom one ofN-α(2,4,6-triisopropylphenylsulfonyl)-3-amidino-phenylalanine-4-ethoxy-carbonylpiperazide-hydrochlorideor its prodrugN-α-(2,4,6-triisopropylphenylsulfonyl)-3-hydroxyamidino-phenylalanine-4-ethoxycarbonylpiperazide,wherein the selected compound can be present as (L)- or (D)-enantiomers,and as E- or (Z)-isomers or (E/Z)-mixtures, and as free bases or assalts thereof. In an embodiment, the method further comprisesdiagnostically confirming that the subject is infected with SARS-CoV-2prior to administering the compound. In an embodiment, the total dose ofthe compoundN-α-(2,4,6-triisopropylphenylsulfonyl)-3-hydroxyamidino-phenylalanine-4-ethoxycarbonylpiperazideper day is independently selected upon each occurrence from about 200 mgto about 400 mg. In an embodiment, the compound isN-α-(2,4,6-triisopropylphenylsulfonyl)-3-hydroxyamidino-(L)-phenylalanine-4-ethoxycarbonylpiperaziniumhydrogen sulfate.

According to aspects illustrated herein, there is disclosed a method fortreating COVID-19 (SARS-CoV-2) coronavirus infection, comprisingadministering to a human subject in need thereof a therapeuticallyacceptable amount of a compound selected from one ofN-α(2,4,6-triisopropylphenylsulfonyl)-3-amidino-phenylalanine-4-ethoxy-carbonylpiperazide-hydrochlorideor its prodrugN-α-(2,4,6-triisopropylphenylsulfonyl)-3-hydroxyamidino-phenylalanine-4-ethoxycarbonylpiperazide,wherein the selected compound can be present as (L)- or (D)-enantiomers,and as E- or (Z)-isomers or (E/Z)-mixtures, and as free bases or assalts thereof, the compound having the ability to bind a hemagglutinin(HA)-activating type II transmembrane serine proteases (TTSPs), therebydecreasing coronavirus replication in the human subject followingexposure to coronavirus. In an embodiment, the TTSP is transmembraneprotease serine SI member 2 (TMPRSS2). In an embodiment, the TTSP istransmembrane protease serine 11A (TMPRSS11(A)). In an embodiment, themethod further comprises diagnostically confirming that the subject isinfected with SARS-CoV-2 prior to administering the compound. In anembodiment, the compound isN-α-(2,4,6-triisopropylphenylsulfonyl)-3-hydroxyamidino-(L)-phenylalanine-4-ethoxycarbonylpiperaziniumhydrogen sulfate.

According to aspects illustrated herein, there is disclosed a method ofmodulating replication of coronavirus in a host cell infected with thecoronavirus comprising administering to the host cell a compoundselected from one ofN-α(2,4,6-triisopropylphenylsulfonyl)-3-amidino-phenylalanine-4-ethoxy-carbonylpiperazide-hydrochlorideor its prodrugN-α-(2,4,6-triisopropylphenylsulfonyl)-3-hydroxyamidino-phenylalanine-4-ethoxycarbonylpiperazide,wherein the selected compound can be present as (L)- or (D)-enantiomers,and as E- or (Z)-isomers or (E/Z)-mixtures, and as free bases or assalts thereof, in an amount effective to modulate replication of thevirus. In an embodiment, the compound isN-α-(2,4,6-triisopropylphenylsulfonyl)-3-hydroxyamidino-(L)-phenylalanine-4-ethoxycarbonylpiperaziniumhydrogen sulfate.

According to aspects illustrated herein, there is disclosed use ofWX-671 in the preparation of drugs for treating coronavirus infection.In an embodiment, the coronavirus is a 2019 novel coronavirus COVID-19.In an embodiment, the coronavirus infection is coronavirus pneumonia. Inan embodiment, the WX-671 is active against a host serine proteaseinhibitor and blocks the spike protein-driven entry into host cells.

According to aspects illustrated herein, the present invention featuresa packaged pharmaceutical product. The packaged pharmaceutical productincludes a container, a plurality of WX-671 unit dosage forms suitablefor oral administration in the container, and a legend (e.g., a label oran insert) associated with the container and indicating administrationof WX-671 for treating 2019 coronavirus disease (COVID-19) caused by theSARS-CoV-2 virus.

BRIEF DESCRIPTION OF THE DRAWINGS

The presently disclosed embodiments will be further explained withreference to the attached drawings. The drawings shown are notnecessarily to scale, with emphasis instead generally being placed uponillustrating the principles of the presently disclosed embodiments.

FIG. 1 is a curve fitting equation 1 with the fractional velocity on they-axis and WX-UK1 concentration on the x-axis. The graph shows howWX-UK1 inhibits the activity of TMPRSS2

FIG. 2 is a curve fitting equation 1 with the fractional velocity on they-axis and WX-UK1 concentration on the x-axis. The graph shows howWX-UK1 inhibits the activity of TMPRSS11A.

FIG. 3 is a depiction of the human EpiAirway™ cell culture model, hereinreferred to as human bronchial epithelial cells (HBEC).

FIG. 4A is a graph showing that in WX-UK1-treated and upamostat-treated,SARS-CoV-2 infected HBEC cultures, after 3 days incubation, adose-dependent reduction in infectious virus production was observed atpharmacologically relevant concentrations. The virus was titered viaTCID50 assay in apical washes. Each symbol represents the titer,averaged from 3 replicates tested.

FIG. 4B is a graph showing that in upamostat-treated, SARS-CoV-2infected HBEC cultures, after 3 days incubation, a dose-dependentreduction in infectious virus production was observed atpharmacologically relevant concentrations. The virus was titered viaplaque reduction assay in apical washes. Each symbol represents thetiter, averaged from 3 replicates tested.

FIG. 5 is a graph showing that in WX-UK1-treated and upamostat-treated,SARS-CoV-2-infected HBEC cultures, after 3 days incubation, limitedcytotoxicity across the dose range where the potent anti-viral effectsare seen.

FIG. 6A and FIG. 6B are graphs demonstrating the inhibition by upamostatand WX-UK1 of SARS-2-S-driven entry in Calu-3 cells and Vero-E6 Cells.FIG. 6A Calu-3 cells or FIG. 6B Vero-E6 cells were pre-incubated withthe indicated concentrations of upamostat, WX-UK1, camostat mesylate, orchloroquine and subsequently inoculated with pseudoparticles harboringthe VSV-SARS-2 S protein. Pseudotype entry was analyzed by determiningluciferase activity in cell lysates. The results of a single experimentperformed with quadruplicate samples are shown. Error bars indicatestandard deviation (SD).

FIG. 7 is a graph demonstrating the inhibition by upamostat and WX-UK1of VSV-g driven entry in Calu-3 cells. Calu-3 cells were pre-incubatedwith the indicated concentrations of upamostat, WX-UK1, camostatmesylate, or chloroquine and subsequently inoculated withpseudoparticles harboring the VSV-g protein. Pseudotype entry wasanalyzed by determining luciferase activity in cell lysates. The resultsof a single experiment performed with quadruplicate samples are shown.Error bars indicate standard deviation (SD).

DEFINITIONS

As used herein, the term “agent” refers to a drug substance havingpharmacological activity—an effect of the agent on an individual. Theterms “agent,” “active ingredient”, “drug substance,” and “compound” areused interchangeably herein.

As used herein, the term WX-671 refers to(N-α-(2,4,6-triisopropylphenylsulfonyl)-3-hydroxyamidino-phenylalanine-4-ethoxycarbonylpiperazide)as (L)- or (D)-enantiomers, and as E- or (Z)-isomers or (E/Z)-mixtures,and as free bases or as salts thereof. In the case of compounds, salts,prodrugs or solvates that are solids, it is understood by those skilledin the art that the inventive compounds, salts, and solvates may existin different crystal forms, all of which are intended to be within thescope of the present invention. WX-671.1 (upamostat) is a specificcrystalline salt form of WX-671.

Amounts and weights mentioned in this disclosure typically refer to thefree form (free base) (i.e., non-salt, hydrate or solvate form). Thetypical values described herein represent free-form equivalents, i.e.,quantities as if the free form would be administered. If salts areadministered the amounts need to be calculated in function of themolecular weight ratio between the salt and the free form. The weight ofactive compound in the dosage form described herein is with respect toeither the free form or the salt form of the compound unless otherwisespecifically indicated. For example, about 231 mg of WX-671.1(upamostat) is the equivalent to approximately 200 mg of WX-671 (thefree form).

As used herein, the term “coronavirus” includes naturally occurring(e.g. wild-type) coronavirus; naturally occurring coronavirus variants;and coronavirus variants generated in the laboratory, including variantsgenerated by selection, variants generated by chemical modification, andgenetically modified variants (e.g., coronavirus modified in alaboratory by recombinant DNA methods). In an embodiment, a subject canbe tested for a viral infection within a few days after symptoms begin,or after treatment according to the present disclosure, by collectingnasal secretions (nasal or nasopharyngeal (NP) swabs), throat(oropharyngeal) swab, blood, or other body fluid samples and testing thesample for detection of viral antigens or RNA in blood and other bodyfluids using, for example, an antigen-capture enzyme-linkedimmunosorbent assay (ELISA), using an IgM ELISA (to determine whetherthe subject has IgM antibodies), using an IgG ELISA (to determinewhether the subject has IgG antibodies), using polymerase chain reaction(PCR), or by virus isolation. In an embodiment, the coronavirus isselected from the group consisting of Middle East respiratory syndrome(MERS), severe acute respiratory syndrome (SARS) and SARS-CoV-2.

The terms “comprise(s),” “include(s),” “having,” “has,” “can,”“contain(s),” and variants thereof, as used herein, are intended to beopen-ended transitional phrases, terms, or words that do not precludethe possibility of additional acts or structures. The singular forms“a,” “and” and “the” include plural references unless the contextclearly dictates otherwise. The present disclosure also contemplatesother embodiments “comprising,” “consisting of” and “consistingessentially of,” the embodiments or elements presented herein, whetherexplicitly set forth or not.

The terms “co-administer,” “coadministration,” or “in combination” areused to describe the administration of a compound of the presentinvention in combination with at least one other antiviral active agent.The timing of the coadministration is best determined by the medicalspecialist treating the patient. It is sometimes desired that the agentsbe administered at the same time. Alternatively, the drugs selected forcombination therapy may be administered at different times to thepatient. Of course, when more than one viral or other infection or othercondition is present, the present compounds may be combined with otheragents to treat that other infection or condition as required.

As related to the present invention, the term “treatment”, “treating”,and the like, is defined as prior to prophylactic administration of thecompounds in the methods described herein, prior to viral infection, orinhibiting viral activity after infection has occurred. In anembodiment, the term “treating” is meant to administer one or morecompounds of the present invention to measurably inhibit the replicationof a virus in vitro or in vivo, to measurably decrease the load of avirus in a cell in vitro or in vivo, or to reduce at least one symptomassociated with having a CoV-mediated disease in a patient. Desirably,the inhibition in replication or the decrease in viral load is at least10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 97%, 98%, 99%, as determined using a suitable assay.Assays that monitor replication of viruses include, but are not limitedto, cytopathic viral assays, reporter-virus and reporter-cell assays,viral replicon assays, and gene-targeted viral assays. Viral loadtesting can be carried out using nucleic acid amplification based tests(NATs or NAATs) and non-nucleic acid-based tests on blood plasma samplesto determine the quantity of virus in a given volume including viral RNAlevels in plasma and tissue and total viral DNA. Alternatively, incertain embodiments, treatment is observed by a trained physician as anappreciable or substantial relief of symptoms in a patient with aCoV-mediated disease. Typically, a decrease in viral replication isaccomplished by reducing the rate of RNA polymerization, RNAtranslation, protein processing or modification, or by reducing theactivity of a molecule involved in any step of viral replication (e.g.,proteins or coded by the genome of the virus or host important for viralreplication). In an embodiment, the term “treat” refers to the abilityof a compound or compounds of the present invention to inhibit orsuppress replication of a virus, such as an RNA virus. In an embodiment,the term “treat” refers to the ability of a compound or compounds of thepresent invention to inhibit the cytopathic effect during a RNA virusinfection.

In some embodiments, an “effective amount” or “immune-stimulatoryamount” of a compound of the invention is an amount which, whenadministered to a subject, is sufficient to engender a detectable immuneresponse. In other embodiments, a “protective effective amount” of animmunogenic composition is an amount which, when administered to asubject, is sufficient to confer protective immunity upon the subject.In other embodiments, a “therapeutic effect amount” of a compound is anamount which, when administered to a subject, is sufficient to treat aviral infection, such as increase viral clearance.

The agents and methods of the present invention may be utilized to treata subject in need thereof. In certain embodiments, the subject is amammal such as a human, or a non-human mammal. When administered to ananimal, such as a human, the agent is preferably administered as apharmaceutical composition comprising, for example, at least one agentof the invention with a substance or collection of substances capable ofbeing combined with the at least one agent. The term“pharmaceutically-acceptable carrier materials” as used herein means asubstance or collection of substances capable of being combined with anagent that is suitable for use in contact with the tissues of mammalsfor purposes of a therapeutic treatment in the mammals under anticipatedexposure conditions. Pharmaceutically-acceptable carrier materials arewell known in the art and include, for example, inert solid, semi-solidor liquid filler, diluent, encapsulating material.Pharmaceutically-acceptable carrier materials must, of course, be ofsufficiently high purity and sufficiently low toxicity to render themsuitable for administration to the human or lower animal being treated.The pharmaceutical composition can be in unit dosage form such astablet, capsule (including sprinkle capsule and gelatin capsule),granule, powder, syrup, suppository, injection or the like.

The term “immune response” refers to a response of a cell of the immunesystem, such as a B-cell, T-cell, macrophage or polymorphonucleocyte, toa stimulus such as an antigen. An immune response can include any cellof the body involved in a host defense response, including for example,an epithelial cell that secretes an interferon or a cytokine. An immuneresponse includes, but is not limited to, an innate immune response orinflammation. As used herein, a protective immune response refers to animmune response that protects a subject from infection (preventsinfection or prevents the development of disease associated withinfection).

By “more effective” is meant that a treatment exhibits greater efficacy,or is less toxic, safer, more convenient, or less expensive than anothertreatment with which it is being compared. Efficacy may be measured by askilled practitioner using any standard method that is appropriate for agiven indication.

As used herein, the term “a suitable period of time” refers to theperiod of time starting when a patient begins treatment for a diagnosisof coronavirus infection using a method of the present disclosure,throughout the treatment, and up until when the patient stops treatmentdue to either a reduction in symptoms associated with the coronavirusinfection or due to a laboratory diagnosis indicating that the viralinfection is under control. In an embodiment, a suitable period of timeis one (1) week. In an embodiment, a suitable period of time is betweenone (1) week and two (2) weeks. In an embodiment, a suitable period oftime is two (2) weeks. In an embodiment, a suitable period of time isbetween two (2) weeks and three (3) weeks. In an embodiment, a suitableperiod of time is three (3) weeks. In an embodiment, a suitable periodof time is between three (3) weeks and four (4) weeks. In an embodiment,a suitable period of time is four (4) weeks. In an embodiment, asuitable period of time is between four (4) weeks and five (5) weeks. Inan embodiment, a suitable period of time is five (5) weeks. In anembodiment, a suitable period of time is between five (5) weeks and six(6) weeks. In an embodiment, a suitable period of time is six (6) weeks.In an embodiment, a suitable period of time is between six (6) weeks andseven (7) weeks. In an embodiment, a suitable period of time is seven(7) weeks. In an embodiment, a suitable period of time is between seven(7) weeks and eight (8) weeks. In an embodiment, a suitable period oftime is eight (8) weeks.

As used herein, the term “cytopathic effects” refers to the changes incell morphology due to a viral infection.

As used herein, the terms “cytopathogenesis” or “pathogenesis” includesinhibition of host cell gene expression and includes other cellularchanges that contribute to viral pathogenesis in addition to thosechanges that are visible at the microscopic level.

As used herein, the term “inhibitor” refers to a molecule that affectsthe activity of enzymes. The inhibitors of the present invention arereversible meaning they form weak interactions with their target enzymeand are easily removed. A reversible inhibitor forms a transientinteraction with an enzyme. The strength of the binding between anenzyme and a reversible inhibitor is defined by the dissociationconstant (K_(d)). The smaller the value of K_(d) the stronger theinteraction between the enzyme and inhibitor and the greater theinhibitory effect. When talking about enzyme inhibition K_(d) isreferred to as K_(i).

The term “in vitro” as used herein refers to procedures performed in anartificial environment, such as for example, without limitation, in atest tube or cell culture system. The skilled artisan will understandthat, for example, an isolate SK enzyme may be contacted with amodulator in an in vitro environment. Alternatively, an isolated cellmay be contacted with a modulator in an in vitro environment.

The term “in vivo” as used herein refers to procedures performed withina living organism such as, without limitation, a human, monkey, mouse,rat, rabbit, bovine, equine, porcine, canine, feline, or primate.

DETAILED DESCRIPTION

The disclosure relates generally to the fields of virology, infectiousdisease, and medicine. And describes compounds, compositions, methodsand kits for the treatment of CoV-mediated disease, e.g., one caused bySARS-CoV-2, SARS, or MERS. In an embodiment, the compositions compriseWX-671 and a pharmaceutically-acceptable carrier material. In anembodiment, the present disclosure describes a new use/application ofupamostat in the preparation of medicines for treating coronavirusinfection in humans.

More specifically, the invention relates to effective inhibitors ofcoronaviruses which can treat coronaviruses, including the 2019 novelcoronavirus. The invention provides a new use of upamostat as aneffective inhibitor of coronaviruses, and its application in thepreparation of drugs for treating coronavirus infection in humans.

WX-671, (N-α-(2,4,6-triisopropylphenylsulfonyl)-3-hydroxyamidino-phenylalanine-4-ethoxycarbonylpiperazide), isan orally active prodrug of the potent serine protease inhibitor WX-UK1(N-α-(2,4,6-triisopropylphenylsulfonyl)-3-amidino-phenylalanine-4-ethoxycarbonylpiperazide).WX-671 is represented by the following structural formula:

and can be prepared as (L)- or (D)-enantiomers, and as E- or (Z)-isomersor (E/Z)-mixtures, and as free bases or as salts thereof.

WX-671 is a prodrug. As used herein, a prodrug refers to apharmaceutical composition that includes a biologically inactivecompound that is metabolized in vivo to generate the active form of thedrug. WX-671 is a compound which is convertible in vivo to affordWX-UK1. WX-UK1 can only be administered by intravenous infusion. WX-UK1is used in many of the experimental in vitro examples described herein.While the present disclosure describes the oral WX-671 compound as amedicament, it should be understood that a medicament can be made usingthe intravenous infusion compound WX-UK1, which is within the scope andspirit of the present invention. U.S. Pat. Nos. 6,861,435, 7,247,724,7,659,396, and 9,089,532, which are incorporated herein by reference,disclose WX-UK1 and methods of making same.

WX-671.1,N-α-(2,4,6-triisopropylphenylsulfonyl)-3-hydroxyamidino-(L)phenylalanine-4-ethoxycarbonylpiperazidehydrogen sulfate, also referred to as Ethyl4-{3-[(E)-amino(hydroxyimino)methyl]-N-[(2,4,6-triisopropylphenyl)sulfonyl]-L-phenylalanyl}piperazine-1-carboxylatehydrogen sulfate, has the molecular formula C₃₂H₄₇N₅O₆S x H₂SO₄ and amolecular mass of 727.91 g/mol (free base: 629.83 g/mol). U.S. Pat. Nos.6,624,169, 7,211,670, 7,247,724, 7,342,018, 7,608,623, 7,659,396,7,713,980, 7,745,441, 7,807,681, 7,884,206, 7,951,943, 8,492,385,8,692,761 and RE46424, which are incorporated herein by reference,disclose these compounds, use, and methods of making same. The substanceWX-671.1 has been given an international nonproprietary name (INN) ofupamostat.

The structural formula of WX-671.1 (upamostat) is as follows:

Upamostat is a non-hygroscopic, white to yellowish powder which isfreely soluble in dimethyl sulfoxide and soluble in ethanol. The drugsubstance is very slightly soluble in water or 0.1 M HCl. Solidpreparations for oral administration can be prepared as tablets, pills,powder, granules, capsules and so forth. In an embodiment, these solidpreparations are manufactured by adding at least onepharmaceutically-acceptable carrier material such as starch, calciumcarbonate, sucrose, lactose, or gelatin. In addition, lubricants such asmagnesium stearate, and talc may be used in addition to the typicalexcipients.

In an embodiment, a medicine is prepared by filling upamostat in hardgelatin capsules that further comprise at least one of the followingexcipients: microcrystalline cellulose; hypromellose; ethyl alcoholanhydrous; purified water and magnesium stearate vegetal. In anembodiment, upamostat capsules contain upamostat hydrogen sulphate231.26 mg (equivalent to 200 mg free base). After oral administration,upamostat is converted to the active WX-UK1, which inhibits severalserine proteases. Because upamostat can be provided as an oralformulation, it can obviate the disadvantages associated withintravenous administration of other drugs that might be useful fortreating coronavirus infection.

Upamostat for treating coronavirus infection is generally administeredin an amount ranging from about 200 mg to about 1000 mg per day. In anembodiment, upamostat is administered as one capsule, once per day, fora total daily dose of about 231.26 mg (equivalent to 200 mg free base).In an embodiment, upamostat is administered as two capsules, once perday, for a total daily dose of about 462.52 mg (equivalent to 400 mgfree base). In an embodiment, a patient with a confirmed coronavirusinfection is provided with instructions to take one capsule of upamostateach day (equivalent to 200 mg upamostat free base), for a total of 2consecutive weeks, or 14 consecutive days. In an embodiment, a patientwith a confirmed coronavirus infection is provided with instructions totake two capsules of upamostat each day equivalent to 400 mg upamostatfree base), for a total of 2 consecutive weeks, or 14 consecutive days.

The inventors have discovered the new use of upamostat after a lot ofresearch. Without being bound by theory, it is believed that the serineprotease inhibitor WX-UK1, the active drug of upamostat once upamostatis broken down inside the body, is active against at least one of theserine proteases that appear to be responsible for viral spike (S)protein priming. Use of protease inhibitors such as WX-UK1 (or it'sprodrug WX-671) may therefore be effective in decreasing CoV activationand spread, resulting in an effective preventative and therapeutictreatment. Therefore, WX-UK1 is able to block SARS-2-S-driven entry intocells, and thus, as a result, will inhibit coronavirus replication. Inan embodiment, since infection requires proteolytic activation whichfacilitates interaction of the virus with host cell receptors, thusenhancing infectivity and spread, upamostat of the present invention,when administered at therapeutically effective amounts and for asuitable period of time, will protect against infection by coronavirus.

Provided herein are packaged pharmaceutical products, also known aspharmaceutical kits, that includes a container, a plurality of upamostatdosage forms suitable for oral administration in the container, and alegend (e.g., a label or an insert) associated with the container andindicating administration of upamostat for treating coronavirusinfection. In an embodiment, the legend includes instructions forcarrying out the methods described above and/or how to use the kit.Instructions included in the kit can be affixed as a label to packagingmaterial or can be included as a package insert. While instructions aretypically written or printed materials, they are not limited to such.Any medium capable of storing instructions and communicating them to anend user is contemplated by this disclosure. Such media include, but arenot limited to, electronic storage media (e.g., magnetic discs, tapes,cartridges), optical media (e.g., CD ROM), and the like. As used herein,the term “instructions” can include the address of an internet sitewhich provides instructions.

Combination and Alternation Therapy

The compounds described herein can be administered on top of the currentstandard of care for COVID patients, or in combination or alternationwith any other compound or therapy that the healthcare provider deemsbeneficial for the patient. The combination and/or alternation therapycan be therapeutic, adjunctive, or palliative. When the methods includeadministering to a patient more than one active agent, the agents may beadministered within 7, 6, 5, 4, 3, 2 or 1 days; within 24, 12, 6, 5, 4,3, 2 or 1 hours, within 60, 50, 40, 30, 20, 10, 5 or 1 minutes; orsubstantially simultaneously. The methods of the invention may includeadministering one or more agents to the patient by oral, systemic,parenteral, topical, intravenous, inhalational, or intramuscularadministration.

It has been observed that COVID patients can pass through various stagesof disease, and that the standard of care can differ based on what stageof illness the patient presents with or advances to. COVID is noteworthyfor the development of “cross-talk” between the immune system and thecoagulation system. As the disease progresses, the patient can mount anoverreaction by the immune system, which can lead to a number of seriousimplications, including a cytokine storm. Via the cross-talk between theimmune system and the coagulation system, the patient can begin clottingin various areas of the body, including the respiratory system, brain,heart and other organs. Multiple clots throughout the body have beenobserved in COVID patients, requiring anticoagulant therapy. It isconsidered that these clots may cause long term, or even permanentdamage if not treated and disease alleviated.

More specifically, COVID-19 has been described as progressing throughthree general stages of illness: stage 1 (early infection), stage 2(pulmonary phase), and stage 3 (hyperinflammation phase/cytokine storm).

Stage 1 is characterized by non-specific, and often mild, symptoms.Viral replication is occurring, and it is appropriate to begin immediatetreatment with the compounds described herein and perhaps in combinationor alternation with another anti-viral therapy. Interferon-β may also beadministered to augment the innate immune response to the virus. In oneembodiment, therefore, a compound of the present invention is used in aneffective amount in combination or alternation with interferon-β and oran additional anti-viral drug. Zinc supplements and or Vitamin C is alsosometimes administered at this stage or as the illness progresses.

Stage 2 of COVID-19 is the pulmonary phase where patients may experienceacute hypoxemic respiratory failure. In fact, the primary organ failureof COVID-19 is hypoxemic respiratory failure. It has been shown thatmoderate immunosuppression via a steroid, for example, dexamethasone,can be beneficial to patients with acute hypoxemic respiratory failureand/or patients on mechanical ventilation. In one embodiment, a compoundthe present invention is used in an effective amount in combination witha corticosteroid which may be a glucocorticoid. Non-limiting examplesare budesonide (Entocort EC), bethamethasone, (Celestone), prednisone(Prednisone Intensol), prednisolone (Orapred, Prelone), triamcinolone(Aristospan Intra-Articular, Aristospan Intralesional, Kenalog),methylprednisolone (Medrol, Depo-Medrol, Solu-Medrol), hydrocortisone,or dexamethasone (Dexamethasone Intensol, DexPak 10 Day, DexPak 13 Day,DexPak 6 Day).

The NS5B inhibitor Remdesivir has provided mixed results when given toCOVID19 patients. It can only be administered in a hospital setting, andonly by intravenous injection, typically three times a day, which makesit inappropriate for mild to moderate COVID19 patients. In oneembodiment, a compound of the present invention is administered incombination or in alternation with Remdesivir to amplify the overallantiviral effect.

Stage 3, the final stage of the disease, is characterized by progressivedisseminated intravascular coagulation (DIC), a condition in which smallblood clots develop throughout the bloodstream. This stage also caninclude multi-organ failure (e.g. vasodilatory shock, myocarditis). Ithas also been observed that many patients respond to this severe stageof COVID-19 infection with a “cytokine storm.” There does appear to be abi-directional, synergistic relationship between DIC and cytokine storm.To combat DIC, patients are often administered an anti-coagulant agent,which may, for example, be an indirect thrombin inhibitor or a directoral anticoagulant (“DOAC”). Non-limiting examples are low-molecularweight heparin, warfarin, bivalirudin (Angiomax), rivaroxaban (Xarelto),dabigatran (Pradaxa), apixaban (Eliquis), or edoxaban (Lixiana). In oneembodiment, a compound of the present invention is administered incombination or in alternation with anti-coagulant therapy. In somesevere cases of clotting in COVID patients, TPA can be administered(tissue plasminogen activator).

It has been observed that high levels of the cytokine interleukin-6(IL-6) are a precursor to respiratory failure and death in COVID-19patients. To treat this surge of an immune response, which mayconstitute a cytokine storm, patients can be administered anIL-6-targeting monoclonal antibody, pharmaceutical inhibitor or proteindegrader such as a bispecific compound that binds to IL-6 and also to aprotein that mediates degradation. Examples of antibodies includetocilizumab, sarilumab, siltuximab, olokizumab and clazakizumab. In oneembodiment, a compound of the present invention is administered incombination or in alternation with tocilizumab or sarilumab. Additionalnonlimiting examples of immunosuppressant drugs used to treat theoverreacting immune system include Janus kinase inhibitors (tofacitinib(Xeljanz)); calcineurin inhibitors (cyclosporine (Neoral, Sandimmune,SangCya)), tacrolimus (Astagraf XL, Envarsus XR, Prograf)); mTORinhibitors (sirolimus (Rapamune), everolimus (Afinitor, Zortress)); and,IMDH inhibitors (azathioprine (Azasan, Imuran), leflunomide (Arava),mycophenolate (CellCept, Myfortic)). Additional antibodies and biologicsinclude abatacept (Orencia), adalimumab (Humira), anakinra (Kineret),certolizumab (Cimzia), etanercept (Enbrel), golimumab (Simponi),infliximab (Remicade), ixekizumab (Taltz), natalizumab (Tysabri),rituximab (Rituxan), secukinumab (Cosentyx), tocilizumab (Actemra),ustekinumab (Stelara), vedolizumab (Entyvio), basiliximab (Simulect),and daclizumab (Zinbryta)).

IL-1 blocks the production of IL-6 and other proinflammatory cytokines.COVID patients are also sometimes treated with anti-IL-1 therapy toreduce a hyperinflammatory response, for example, an intravenousadministration of anakinra. Anti-IL-1 therapy generally may be forexample, a targeting monoclonal antibody, pharmaceutical inhibitor orprotein degrader such as a bispecific compound that binds to IL-1 andalso to a protein that mediates degradation.

Patients with COVID often develop viral pneumonia, which can lead tobacterial pneumonia. Patients with severe COVID-19 can also be affectedby sepsis or “septic shock”. Treatment for bacterial pneumonia secondaryto COVID or for sepsis includes the administration of antibiotics, forexample a macrolide antibiotic, including azithromycin, clarithromycin,erythromycin, or roxithromycin. Additional antibiotics includeamoxicillin, doxycycline, cephalexin, ciprofloxacin, clindamycin,metronidazole, sulfamethoxazole, trimethoprim, amoxicillin, clavulanate,or levofloxacin. In one embodiment, thus a compound of the presentinvention, is administered in combination or in alternation with anantibiotic, for example, azithromycin. Some of these antibiotics such asazithromycin have independent anti-inflammatory properties. Such drugsmay be used both as anti-inflammatory agents for COVID patients and havea treatment effect on secondary bacterial infections.

A unique challenge in treating patients infected with COVID-19 is therelatively long-term need for sedation if patients require mechanicalventilation which might last up to or greater than 5, 10 or even 14days. For ongoing pain during this treatment, analgesics can be addedsequentially, and for ongoing anxiety, sedatives can be addedsequentially. Non-limiting examples of analgesics include acetaminophen,ketamine, and PRN opioids (hydromorphone, fentanyl, and morphine).Non-limiting examples of sedatives include melatonin, atypicalantipsychotics with sedative-predominant properties (olanzapine,quetiapine), propofol or dexmedetomidine, haloperidol, andphenobarbital. In one embodiment, a compound of the present invention isadministered in combination or in alternation with a pain reliever, suchas acetaminophen, ketamine, hydromorphone, fentanyl, or morphine. In oneembodiment, a compound of the present invention is administered incombination or in alternation with a sedative, such as melatonin,olanzapine, quetiapine, propofol, dexmedetomidine, haloperidol, orphenobarbital.

Investigational drugs for COVID-19 include chloroquine andhydroxychloroquine. In one embodiment, a compound of the presentinvention, is administered in combination or in alternation withchloroquine or hydroxychloroquine.

A protease inhibitor such as lopinavir or ritonavir, previously approvedfor HIV, may also be administered.

Additional drugs that may be used in the treatment of a COVID patientinclude, but are not limited to favipiravir, fingolimod (Gilenya),methylprednisolone, bevacizumab (Avastin), Actemra (tocilizumab),umifenovir, losartan and the monoclonal antibody combination of REGN3048and REGN3051 or ribavirin. Any of these drugs or vaccines can be used incombination or alternation with an active compound provided herein totreat a viral infection susceptible to such.

In one embodiment, a compound of the present invention is used in aneffective amount in combination with anti-coronavirus vaccine therapy,including but not limited to mRNA-1273 (Moderna, Inc.), AZD-1222(AstraZeneca and University of Oxford), BNT162 (Pfizer and BioNTech),CoronaVac (Sinovac), NVX-CoV 2372 (NovoVax), SCB-2019 (Sanofi and GSK),ZyCoV-D (Zydus Cadila), and CoVaxin (Bharat Biotech). In anotherembodiment, a compound of the present invention is used in an effectiveamount in combination with passive antibody therapy or convalescentplasma therapy.

In an embodiment, a compound of the present invention is used in aneffective amount in combination with a 5-HT receptor antagonists, whichcan relieve certain symptoms that might be present in a patient infectedwith coronavirus, such as diarrhea.

SARS-CoV-2 is constantly mutating, which many increase virulence andtransmission rates. Drug-resistant variants of viruses may emerge afterprolonged treatment with an antiviral agent. Drug resistance may occurby mutation of a gene that encodes for an enzyme used in viralreplication. The efficacy of a drug against an RNA virus infection incertain cases can be prolonged, augmented, or restored by administeringthe compound in combination or alternation with another, and perhapseven two or three other, antiviral compounds that induce a differentmutation or act through a different pathway, from that of the principledrug.

The present invention has multiple aspects, illustrated by the followingnon-limiting examples. The following examples are given for the purposeof illustrating various embodiments of the invention and are not meantto limit the present invention in any fashion.

EXAMPLES Example 1: Evaluation of TMPRSS2 and TMPRSS11(A) as Targets forWX-UK1 Inhibition

Several enzymes, pertaining to different protease families, can behijacked by CoV S proteins for priming. The pH-dependent cysteineprotease cathepsin L, TMPRSS2, TMPRSS11A, as well as the serine proteasefurin can prime S proteins during viral entry into target cell. Weperformed an analysis, including structure modelling/prediction,structure analysis and review of relevant literature to determine if anyof the TTSPs are a relevant target of inhibition by upamostat.

TMPRSS2 and TMPRS11A mammalian expression systems were purchased fromMyBioSource (MBS1193731 and MBS1345824, respectively). Proteins werereconstituted to 1 mg/ml according to the manufacturer, and we ran a gelwith the reconstituted proteins. A fresh stock solution of WX-UK1 wasmade (100 mM WX-UK1 in 100% DMSO). Concentrated stock was diluted to 1mM in HBS buffer before further dilution in assays.

Enzyme inhibitors may interact with enzymes and/or enzyme-substratecomplexes in several different ways to diminish the rate of anenzyme-catalyzed reaction. For each mode of inhibition, one cancalculate a dissociation constant, Ki, for the inhibitor that reflectsthe strength of the interaction between the enzyme and the inhibitor. Kifor an inhibitor is analogous to Km for a substrate; a small Ki valuereflects tight binding of an inhibitor to an enzyme, whereas a larger Kivalue reflects weaker binding. The precise formula that is used tocalculate Ki depends on the mode of inhibition, which can be determinedexperimentally by comparing the “apparent” values of V_(max) and Km foran enzyme in the presence of an inhibitor to the V_(max) and Km valuesin the absence of any inhibitor (Equation 2 below).

The chromogenic substrate chosen for these studies was S-2288 substrate.K_(i)-values were determined by measuring the effect of WX-UK1 on humanserine protease cleavage of chromogenic substrates. For determination ofK_(i)-values, concentration series of WX-UK1 were pre-incubated with thetarget human serine protease before chromogenic substrate was added toinitiate the reaction. The reaction velocities were determined from theslopes using linear regression and these were normalized to that of thenon-inhibited reaction. The normalized activities were plotted againstWX-UK1 concentrations before the K_(i)-values were obtained bynon-linear regression using equation 1.

$\begin{matrix}{\frac{v_{i}}{v_{0}} = \frac{{K_{i} \cdot {KM}} + \lbrack S\rbrack}{\left( {K_{i} \cdot \lbrack S\rbrack} \right) + {{KM} \cdot \left( {K_{i} + \lbrack I\rbrack} \right)}}} & {{Equation}\mspace{14mu} 1}\end{matrix}$

vi/v0 is the ratio of initial velocity with and without inhibitor, whichis described as a function of inhibitor concentration, [I] and substrateconcentration, [S].

The K_(M)-parameter was obtained by standard Michaelis-Menten kinetics.Serine protease was added to a suitable concentration series ofsubstrate, high enough to yield an experimental Vmax value. Thesubsequent reaction velocities were plotted against the substrateconcentrations before the KM-value was derived using theMichaelis-Menten equation (2).

$\begin{matrix}{v = \frac{v_{\max} \cdot \lbrack S\rbrack}{K_{M} + \lbrack S\rbrack}} & {{Equation}\mspace{14mu} 2}\end{matrix}$

All experiments were performed in at least triplicates at 37° C. in FIBS(30 mM Hepes, pH=7.4; 150 mM NaCl; 0.5% BSA). Reactions were monitoredat 2 reads/min for at least 45 min at 405 nm. Since WX-UK1 was kept in100% DMSO, an uninhibited DMSO-control was included in all experimentsto exclude unwanted DMSO effects on protease activity.

Inhibition of Human TMPRSS2 with WX-UK1

FIG. 1 is a curve fitting equation 1 with the fractional velocity on they-axis and WX-UK1 concentration on the x-axis. The graph shows howWX-UK1 inhibits the activity of TMPRSS2. The K_(i) was determined to be2.9±0.04 (3) μM.

Inhibition of Human TMPRSS11a with WX-UK1

FIG. 2 is a curve fitting equation 1 with the fractional velocity on they-axis and WX-UK1 concentration on the x-axis. The graph shows howWX-UK1 inhibits the activity of TMPRSS11A. The K_(i) was determined tobe 0.39±0.01 (3) μM.

TABLE 1 lists inhibition constants, Ki values, of WX-UK1 against a panelof proteases: K_(i) (μM) Mean ± SD Protease (n) Human Trypsin-3 0.019 ±0.004 (6) Human Trypsin-2 0.075 ± 0.003 (6) Human Trypsin-6 0.10 ± 0.01(4) Human Trypsin-1 0.19 ± 0.01 (3) Human Matriptase-1 0.20 ± 0.01 (3)Human TMPRSS11(A) 0.39 ± 0.01 (3) Human HATL5 0.7 ± 0.1 (3) HumanEnterokinase 0.71 ± 0.04 (4) Human Thrombin 0.8 ± 0.1 (3) Human uPA 0.9± 0.1 (3) Human FXIa 0.9 ± 0.1 (3) Human two-chain tPA 1.4 ± 0.1 (3)Human HAT 1.5 ± 0.1(6) Human Plasmin 2.4 ± 0.3 (4) Human FIXa 2.5 ± 0.2(3) Human Fxa 2.6 ± 0.4 (3) Human TMPRSS2  2.9 ± 0.04 (3) Human C1s 3.1± 0.4 (5) Human Activated Protein C 3.9 ± 0.2 (3) Human Hepsin 4.3 ± 0.5(5) Human Matriptase-2 6.4 ± 0.3 (4) Human Spinesin 7.7 ± 0.5 (3) HumanTryptase-ε 11 ± 2 (3)  Human DESC-1 13 ± 2 (3)  Human PRSS27 (IC50) 19 ±4 (3)  Human Plasma Kallikrein 26 ± 1 (3)  Human HGFA 28 ± 5 (4)  HumanGranzyme A >250 (3) Human Kallikrein-8 >250 (3) Human Kallikrein-1 >250(3) Human Kallikrein-11 >250 (3) Human Prostasin (IC50) >250 (3) Rat uPA0.4 ± 0.1 (3) Bovine Cationic Trypsin-1 0.5 ± 0.1 (6) Canine uPA 0.7 ±0.1 (4) Rabbit uPA 0.8 ± 0.1 (3) Human uPA (Q192A in 2.9 ± 0.1 (3)medium) Human uPA (H99A in  14 ± 0.4 (3) medium) Mouse uPA 45 ± 6 (3) 

Example 2: Assessment of the Anti-Viral Activity of Upamostat and WX-UK1Against SARS-CoV-2 in Human Airway Epithelial Cells

We designed an in vitro assessment in an organotypicair-liquid-interface (ALI) culture of human primary bronchial epithelialcells (HBEC; EpiAirway™, MatTek) to evaluate whether infection andspread of SARS-CoV-2 could be directly inhibited by upamostat andWX-UK1. This human cell culture model system was selected because itcontains a pseudostratified epithelial layer that morphologically andfunctionally resembles that of the human airway, consisting of ciliatedand goblet (mucus producing) cells exposed to the air from the apicallayer. These cells act as the first line of defense against invadingviruses and serve as replication sites. Available evidence also suggeststhat human bronchial epithelial cells express host factors targeted byupamostat (e.g., TMPRSS2).

Test Compounds:

Upamostat—Test Compound

-   -   Description: Upamostat—ethyl        4-{3-[(E)-amino(hydroxyimino)methyl]-N-[(2,4,6-triisopropylphenyl)        sulfonyl]-L-phenylalanyl}-piperazine-1-carboxylate hydrogen        sulphate.    -   Solvent: DMSO        WX-UK1—Test Compound    -   Description: WX-UK1—ethyl        4-[(25)-3-(3-carbamimidoylphenyl)-2-[(2,4,6-triisopropylphenyl)sulfonylamino]propanoyl]piperazine-1-carboxylate.    -   Solvent: DMSO        Camostat Mesylate—Test Compound    -   Description: Camostat mesylate (CM)        4-[[4-[(Aminoiminomethyl)amino]benzoyl]oxy] benzeneacetic acid        2-(dimethylamino)-2-oxoethyl ester methanesulfonate; FOY 305;        FOY-S 980; Foipan mesylate. Camostat is a synthetic, orally        bioavailable serine protease.    -   Solvent: DMSO        Bleomycin (Sulfate)—Positive Cytotoxic Control    -   Description: Bleomycin is a chemotherapy agent commonly used for        the treatment of Hodgkin's lymphoma and embryonal carcinomas. A        broad spectrum of bleomycin-induced pulmonary toxicities have        been well described as a complication of such therapy, the most        common variant of which is bleomycin-induced pneumonitis (BIP)        (Sleijfer et al., 2001). Bleomycin (BLM) is chosen as the        best-studied micronucleus (MN) inducers in human lymphocytes        with different mechanisms of genotoxicity.    -   Solvent: DMSO 16.67 mg/mL (11.2 mM)        Methods:        Cell Culture—Differentiated Human Bronchial Epithelial Cells        (HBEC)

Normal human bronchial epithelial (HBEC) cells were differentiated byMatTek Corporation (Ashland, Mass.) and arrived in kits with either 12-or 24-well inserts each. HBEC cells were grown on 6 mm{circumflex over( )}2 mesh disks in transwell inserts. Three days prior to shipment, thetissues were transferred into hydrocortisone-free medium. Duringtransportation the tissues were stabilized on a sheet of agarose, whichwas removed upon receipt. One insert was estimated to consist ofapproximately 1.2×10⁶ cells. Kits of cell inserts (EpiAirway™ AIR-100)originated from a single donor, #9831, a 23-year old, healthy,non-smoking, Caucasian male. The cells have unique properties in forminglayers, the apical side of which is exposed only to air and that createsa mucin layer. Upon arrival, the cell transwell inserts were immediatelytransferred to individual wells of a 6-well plate according tomanufacturer's instructions, and 1 mL of MatTek's proprietary culturemedium (AIR-100-MM) was added to the basolateral side, whereas theapical side was exposed to a humidified 5% CO2 environment. Cells werecultured at 37° C. for one day before the start of the experiment. Afterthe 16-18 h equilibration period, the mucin layer, secreted from theapical side of the cells, was removed by washing with 400 μL pre-warmedTEER buffer. Culture medium was replenished following the wash step. Adepiction of the culture inserts and EpiAirway tissue provided in FIG.3.

Treatment with Test Compounds:

Test compounds were serially diluted from stock solution (containingDMSO) in Assay medium (AIR-ASY-100, MatTek) and placed at roomtemperature. Test compound dilutions are outlined below (final DMSO<0.5%). HBEC cultures were washed with phosphate-buffered saline (PBS)and incubated at 37° C. with Bleomycin sulfate (75.6 and 151 μg/ml),upamostat (6 concentrations ranging from 0.12 to 30.00 μg/ml), WX-UK1(3.33, 10, and 30.00 μg/ml) or camostat (0.5, 5, and 25 μg/ml) dilutedin assay medium (AIR-100-ASY, MatTek) for 1 h prior to infection. Forcontrol wells, assay medium with DMSO (final DMSO <0.5%; control) andvirus only control (assay medium only) were added for the 1 h beforeinfection. Compounds were added to each insert on the apical layer (0.15mL) and basal layer (0.85 mL) in triplicate.

Viral Infection and Sample Processing:

After 1 hr incubation with compounds, the apical side of the cultureswere washed and then infected with SARS-CoV-2 clinical isolate(2019-nCoV/USA-WA1/2020) at MOI=0.1 PFU/cell for 1 h at 37° C., in thepresence of compound or assay control media. After 1 hr viralincubation, the virus was removed from the apical side, and cultureswere washed one time with PBS to remove any unbound virus. The cultureswere then incubated at 37° C. for 72 h with fresh compound. At 24 h and48 h post-infection, the basolateral medium was replaced with 1 mL offresh medium containing the respective compounds.

At 72 hours post-infection, tissues and media were collected forprocessing. The apical layer was washed with 0.4 mL of TEER buffer (PBSwith Mg²⁺ and Ca²⁺) and collected for viral titer assessment via TCID50(50% tissue culture infectious dose) assay. Eight-fold serial dilutionsof apical layer supernatant sample concentrations were added to 96-wellassay plates containing Vero E6 cells (20,000/well). The plates wereincubated at 37° C., 5% CO2 and 95% relative humidity. Following 3 days(72±4 h) incubation, the plates were stained with crystal violet tomeasure cytopathic effect (CPE). Virus titers were calculated using themethod of Reed and Muench (Reed et al., 1938). The TCID50 values weredetermined from triplicate samples. To confirm results from the TCID50assay, a plaque reduction assay was performed. Briefly, 10-fold serialdilutions of apical layer supernatant sample concentrations were addedto 24-well assay plates containing VeroE6 cell (100,000 cells/well) forplaque reduction assay. The plates were incubated at 37° C., 5% CO2 and95% relative humidity. Following 3 days (72±4 h) incubation, the plateswere fixed with 5% neutral buffered formalin and stained with crystalviolet to visualize plaques. The titer was calculated in PFU/mL usingthe following formula: Titer (PFU/mL)=number of plaquescounted×10{circumflex over ( )}^(dilution counted)×10 (to get to mLbecause we added 100 μL of diluted sample). The assay was performedtwice, with a second assay being conducted on virus+DMSO and 0.2 ug/mlupamostat to evaluate additional sample dilutions.

To evaluate the health of HBEC cells after exposure to opaganib, controlcompounds, and viral infection, a Lactate dehydrogenase (LDH) releaseassay was conducted. Medium from the basolateral layer of the tissueculture inserts was removed 72 hours post-infection and diluted in LDHStorage Buffer as per the manufacturer's instructions (Promega). Sampleswere further diluted with LDH Buffer and incubated with an equal volumeof LDH Detection Reagent. Luminescence was recorded after 60 minutesincubation at room temperature. A no cell control was included as anegative control to determine culture medium background and bleomycinincluded as a positive cytotoxic control. Luminescence was reported,with background levels found within the acceptable luminescence range(range 1,000-10,000).

Additionally, the apical layer of the HBEC tissues were collected byadding Trizol LS (Invitrogen) to each culture insert and pipetting upand down several times to lyse and collect the cells and store at −80°C. for future RNA and protein expression analysis.

Results:

Upamostat and WX-UK1, are Highly Potent Antiviral Inhibitors ofSARS-CoV-2 in Human Bronchial Epithelial Tissue Cultures.

In this study, normal human bronchial epithelial cells (HBEC) werepretreated in triplicate with 6 different concentrations of upamostat(ranging from 0.12 to 30.0 μg/ml) and 3 different concentrations ofWX-UK1 (ranging from 3.33 to 30.0 μg/ml) both on the apical andbasolateral side of each culture. Once pretreated, HBEC were exposed toSARS-CoV-2 (2019-nCoV/USA-WA1/2020) and incubated for 3 days withcompound. At 3 days post infection, the apical layer was washed andassessed for viral load by TCID50 assay. The basolateral media wascollected and assessed for presence of lactate dehydrogenase (LDH),which is released from damaged cells serving as an indicator of celldeath/viability. For comparison, 3 concentrations of camostat (rangingfrom 0.5 to 25.0 μg/ml), an established TMPRSS2 inhibitor was included.

Both upamostat and WX-UK1 demonstrated potent antiviral activity, withreplication being inhibited in a dose-dependent manner withoutsignificant compromise to cell viability (except for at the highest doseof each compound). A 3-log and 4-log reduction in viral load wasobserved by TCID50 at the lowest concentration of upamostat (0.12 μg/ml)and WX-UK1 (3.33 μg/ml), respectively. Both upamostat and WX-UK1 sawsimilar reduction in viral titer at 3 days post infection. Cellviability, as assessed in the LDH release assay, was reporteduncompromised at all but the maximum concentrations evaluated forupamostat and WX-UK1.

To demonstrate the anti-viral activity of upamostat and WX-UK1 againstSARS-CoV-2 in a human primary epithelial culture system, we performedanti-viral assays in HBEC cultures, which are grown on air-liquidinterface and recapitulate the cellular complexity and physiology of thehuman conducting airway. In upamostat- and WX-UK1-treated,SARS-CoV-2-infected HBEC cultures, after 3 days incubation, adose-dependent reduction in infectious virus production, confirmed viaTCID50 and plaque reduction assay, were observed at pharmacologicallyrelevant concentrations (FIG. 4A and FIG. 4B). These results comparefavorably with camostat, a known TMPRSS2 inhibitor.

We calculated an EC50 estimate with the plaque reduction assay result.At the highest concentration tested, inhibition of virus productionexceeded 50%. Using graphpad, the EC50 was estimated with the availabledata. The estimated EC50 was 0.02 ug/ml. We utilized the followingformula to calculate % inhibition after converting the TCID values toestimated PFU values as described below:% inhibition=((Value−Avg virus ctrl)/(Avg Cell Ctrl−Avg virus ctrl)*100)The % inhibition values were then analyzed via GraphPad following theseinstructions:

-   -   The X values are upamostat concentrations.    -   The Y values are responses.    -   Selecting “Dose vs. response curve”    -   Selecting Analyze, nonlinear regression, and with the        dose-response (stimulation) set of equations and chose [Dose]        vs. response—variable slope. All other defaults were accepted.        The resulting EC50 was calculated to be 0.02 ug/ml.

Viral replication was inhibited by upamostat, WX-UK1, and camostatwithout significant compromise to cell viability (except at the highestconcentration tested) measured through LDH release. To measure LDHreleased from non-viable cells, medium from the basolateral layer of thetissue culture inserts was removed 72 hours post-infection and dilutedin LDH Storage Buffer as per the manufacturer's instructions (Promega).Samples were further diluted with LDH Buffer and incubated with an equalvolume of LDH Detection Reagent. Luminescence was recorded after 60minutes incubation at room temperature. A no cell control was includedas a negative control to determine culture medium background as well asa positive cytotoxicity control, bleomycin (151 ug/ml). Data are plottedusing the luminescence values minus the no cell control (averageluminescence reading of 6936). This data, using a physiologicallyrelevant human respiratory tissue model, demonstrates upamostat'spotential to strongly inhibit SARS-CoV-2 viral replication, with limitedcytotoxicity in HBEC cultures across the dose range where the potentanti-viral effects are seen (FIG. 5), further demonstrating upamostat'spromising potential for treating patients with COVID-19. The cytotoxicconcentration for 50% of the cell culture (CC50) was generated with theavailable data to determine the compound concentration required toreduce the absorbance of treated cells by 50% in comparison to controlcells. The calculated CC50 value for upamostat, using luminescence datagenerated via the MTT assay, was 46.37 uM (or 29.2 ug/ml). At this CC50concentration, an EC50 concentration lower than 4.6 uM (or 2.9 ug/ml)would result in an SI value (CC50/EC50) >10.

Example 3: Assessment of the Effects of Upamostat and WX-UK1 onSARS-CoV-2 Spike Protein-Mediated Entry

A study was performed to evaluate the inhibitory effects of bothupamostat and WX-UK1 against cellular entry of replication defective,single cycle vesicular stomatitis virus (VSV) particles pseudotyped withthe SARS-CoV-2 spike protein (VSVpp+SARS-2-S Δ18) or the glycoprotein ofvesicular stomatitis virus (VSV) as control. Δ18 refers to the deletionof the 18 C-terminal amino acids of the S protein, which increasespseudotyping efficiency without affecting ACE2 or TMPRSS2 usage.Pseudotype entry and its inhibition was evaluated in Calu-3 and Vero-E6cells. Calu-3 cells, which are a lung-derived human cancer cell line,allow for SARS-CoV-2 S-driven entry in a TMPRSS2-dependent manner.Agents that inhibit TMPRSS2, including camostat, a known TMPRSS2inhibitor shown to inhibit SARS-CoV-2 infection of cultured lung cells(Hoffmann et al., 2020), are expected to inhibit S-driven entry in thismodel. Vero cells, which are a green monkey kidney cell line, permitSARS-CoV-2 spike-driven entry in a TMPRSS2-independent, cathepsinL-dependent manner. Agents that elevate the pH of acidic intracellularendosomes, including chloroquine, are expected to inhibit entry in thismodel. Entry driven by the G-protein of vesicular stomatitis virus (VSV)served as specificity control (VSV-G driven entry depends on low pH andis thus chloroquine but not camostat sensitive).

Methods:

For pseudotyping, vesicular stomatitis virus pseudotype (VSVpp) weregenerated according to a published protocol (Berger Rentsch and Zimmer,2011). In brief, 293T transfected to express the viral surfaceglycoprotein under study were inoculated with a replication-deficientVSV vector that contains expression cassettes for eGFP (enhanced greenfluorescent protein) and firefly luciferase instead of the VSV-G openreadingframe, VSV*DG-fLuc (kindly provided by Gert Zimmer, Institute ofVirology and Immunology, Mittelhaüsern/Switzerland). After an incubationperiod of 1 h at 37 C, the inoculum was removed and cells were washedwith PBS before medium supplemented with anti-VSV-G antibody (Il, mousehybridoma supernatant from CRL-2700; ATCC) was added in order toneutralize residual input virus (no antibody was added to cellsexpressing VSV-G). Pseudotyped particles were harvested 16 hpostinoculation, clarified from cellular debris by centrifugation andused for experimentation.

For transduction, target cells were grown in 96-well plates until theyreached 50%-75% confluency before they were inoculated with respectivepseudotyped. For experiments involving protease inhibitors, target cellswere treated with the respective chemical 2 h before transduction.Transduction efficiency was quantified 16 h posttransduction bymeasuring the activity of firefly luciferase in cell lysates using acommercial substrate (Beetle-Juice, PJK) and a Hidex Senseplateluminometer (Hidex). The transduction assay measures entry of asingle cycle vesicular stomatitis virus (VSV) bearing SARS-CoV-2 spike.

Results:

The ability of upamostat and WX-UK1 to inhibit entry of SARS-2-S andVSV-G bearing pseudotypes was evaluated in Calu-3 (human lung cancercells) and Vero-E6 cells. Calu-3 cells, which are a lung-derived humancancer cell line, allow for SARS-CoV-2 spike-driven entry in aTMPRSS2-dependent manner and thus a camostat-sensitive fashion. Verocells, which are an African green monkey derived kidney cell line,permit SARS-CoV-2 spike-driven entry in a cathepsin L-dependent mannerand chloroquine-sensitive fashion. Entry driven by the G-protein ofvesicular stomatitis virus (VSV) served as specificity control (VSV-Gdriven entry depends on low pH and is thus chloroquine but not camostatsensitive).

Upamostat and WX-UK1 Inhibit SARS-CoV-2 S Protein Mediated Entry inHuman Lung Cancer Cells (Calu-3) and Green Monkey Kidney Cells (Vero E6)with Moderate Efficiency.

When tested against VSVpp+SARS-2-S Δ18 in Calu-3 cells, both WX-UK1 andupamostat showed moderate inhibitory activity, though less thancamostat, another serine protease inhibitor (FIG. 6A). When tested inVero-E6 cells, which do not have surface TMPRSS2, moderate inhibitoryactivity was still noted for upamostat and WX-UK1; camostat was inactivein this situation while the highest concentration of chloroquinepotently inhibited S protein-driven entry (FIG. 6B). WX-UK1 andupamostat moderately inhibited VSV-G entry in Calu-3 cells, suggesting abroader spectrum of activity for upamostat (FIG. 7). All threecompounds, except chloroquine, were inactive when tested against VSV-Gin Vero76 cells. Overall, these results demonstrate WX-UK1 and upamostatinhibit SARS-CoV-2 spike driven entry into Calu-3 and Vero cells withmoderate efficiency. Due to the nature of the model, specificextrapolation to actual in vitro or in vivo inhibitory concentrations isnot possible.

Example 4: Randomized, Double-Blind, Placebo-Controlled Phase 2/3 Studyof Upamostat, a Serine Protease Inhibitor, or Placebo for Treatment ofCOVID-19 Disease

This study will assess the activity of upamostat against placebo fortreatment of COVID-19 patients who, in the investigator's judgment, donot require hospitalization.

Primary Objectives:

Part A of the study: determination of the safety and tolerability of twodose levels and decision regarding upamostat dose for part B. Changes inseverity of disease markers will be assessed, but will not be a primaryfactor in deciding which dose to pursue. Time to recovery will also becalculated, although given the small sample size and expectedvariability of outcome, a clinically meaningful difference may not beseen.

Part B of the study: comparison between upamostat and placebo in time tosustained recovery from illness. A patient will be considered to haverecovered once he or she meets the following criteria:

-   -   1) is afebrile (<38.0° C. core temperature) for at least 48        hours without use of antipyretics;    -   2) all symptoms have resolved or returned to pre-illness levels        (e.g., if patient had baseline respiratory compromise prior to        the onset of COVID-19), except for:        -   a. fatigue, anosmia, ageusia or dysgeusia, which may be            persistent at level similar to that during the acute            illness, i.e., the same level per symptom questionnaire;        -   b. chest pain, cough or dyspnea which if persistent must be            at least one grade lower than at the start of treatment and            no worse than grade 1 (mild).        -   Sustained recovery is recovery, per above definition,            maintained for at least 28 days or through end of study,            whichever comes first.            Secondary Objectives:            Comparison between active treatment group and placebo of:    -   1) Proportion of patients who are PCR-negative at days 8, 15, 29        and 57 from the start of treatment (landmark analyses);    -   2) Time to resolution of individual disease-related symptoms        present at baseline;    -   3) Development of new disease-related symptoms on study;    -   4) Incidence of pneumonia during study among patients without        baseline pneumonia (diagnosed clinically);    -   5) Changes in laboratory markers of disease severity, i.e.,        oxygen saturation, CRP, lymphocyte count, cardiac troponin and        D-dimer levels, from baseline to time points at which these are        measured on study;    -   6) Adverse events;    -   7) Hospitalization within 8 weeks after the first dose of study        medication, overall and for COVID-19-related indications;    -   8) Mortality 30 days after first dose of study medication;        Exploratory    -   1) Percent of patients who report household contacts who have        developed symptomatic, PCR-confirmed, COVID-19 by day 57;    -   2) Levels of serum IgM and IgG antibodies to SARS-CoV-2 at 57        days from the start of treatment.        Safety:        Patients will be followed for adverse events, including both        clinical and laboratory events, throughout the course of the        study.        In particular, toxicities resulting in dose reductions or        discontinuation of therapy will be followed and tabulated.        Population:        Inclusion Criteria:    -   1. Patients with symptomatic, diagnostically confirmed COVID-19,        per RT-PCR assay of respiratory tract sample.    -   2. Patient must have either become symptomatic or found positive        by RT-PCR within 3 days, whichever is greater, of randomization.    -   3. Males and females ≥age 18 years.    -   4. At baseline the laboratory parameters listed below are not        worse than NCI CTCAE v5.0 grade 2, with exceptions noted below:        -   Bilirubin ≤1.5 times upper limit of normal (ULN; grade 1            only)        -   AST (SGOT), ALT (SGPT)≤5.0×ULN,        -   Serum creatinine ≤1.5×ULN (grade 1)        -   Albumin ≥2.0 g/dL    -   5. Acceptable hematologic status:        -   Absolute neutrophil count ≥1000 cells/mm³        -   Platelet count ≥50,000 plt/mm³        -   Hemoglobin ≥8.0 g/dL    -   6. Clinically acceptable blood sugar control in the opinion of        the investigator.    -   7. INR and partial thromboplastin time (PTT) each ≤1.5×ULN        (i.e., grade 1), unless patient is taking dabigatran or heparin.    -   8. Oxygen saturation by pulse oximeter ≥92% on room air    -   9. Negative pregnancy test (if woman of childbearing potential).    -   10. Females of childbearing potential and males with female        partners of childbearing potential must agree to use acceptable        contraceptive methods during the study and for at least two        months after the last dose of study medication.    -   11. Ability to complete the daily diary independently.    -   12. The patient must give informed consent.        Exclusion Criteria:    -   1. Patient is in need of acute hospitalization per clinician        assessment.    -   2. Pregnant or nursing women.    -   3. Unwillingness or inability to comply with procedures required        in this protocol.    -   4. Patient requires supplemental oxygen    -   5. Patient is currently receiving, has received within the past        7 days or is expected to receive during the course of the study        remdesivir, chloroquine, hydroxychloroquine, azithromycin or        other specific antiviral therapy for COVID-19 or systemic        corticosteroid equivalent to ≥0.20 mg daily prednisone/3 mg        dexamethasone daily.    -   6. Patient is currently receiving or has received within 30 days        prior to screening any other investigational agent for any        indication, including approved agents given for investigational        indications (e.g., anti-cytokine treatments).    -   7. Patient is currently taking or is expected to start taking        warafin, apixabain (Eliquis), or rivaroxaban (Xarelto). Patients        may be taking or start on study dabigatran (Pradaxa), standard        or low molecular weight heparin.        Design:

This is a randomized, double-blind, placebo-controlled, parallel groupstudy of upamostat compared to placebo in patients with symptomaticCOVID-19 who do not require inpatient care. The study will use phase 2/3operationally seamless design methodology for dose selection (part A)and inferentially independent confirmatory phase 3 study (part B). Thephase 3 portion will include interim analysis for early termination forfutility or increase in sample size, as indicated by initial results.

Methodology:

Part A: After qualification for study, patients will be stratified byage, <65 or ≥65. They will then be randomized 1:1:1 one of the followingtreatment groups:

-   -   1. Upamostat 200 mg two capsules qd (n=20);    -   2. Upamostat 200 mg one capsule and matching placebo one capsule        qd (n=20)    -   3. Placebo two capsules qd (n=20).        In order to maintain blinding, patients will be given two        bottles of medication and instructed to take one pill from each        bottle each day. Both pills are to be taken at the same time.        Medication should be taken with water and with or without food.        Patients are to take medication for 14 days or until one of the        following occurs:    -   Adverse events, whether related or unrelated to study medication        which, in the judgement of the investigator, necessitate        discontinuation of treatment;    -   The patient or investigator decides that it is in the patient's        best interest to stop treatment.        An interim analysis will be performed by a data safety        monitoring board (DSMB) after a total of 60 patients complete        part A.    -   If the DSMB determines that safety of both regimens is similar,        accrual in part B will continue on the 400 mg qd dose.    -   If safety is more favorable with the 200 mg qd regimen, accrual        in part B will continue on the 200 mg qd dose.        Part B: Based on safety results from part A, either a 200 mg or        400 mg (i.e., one or two 200 mg capsules) treatment regimen will        be selected. Patients enrolled in part B will be stratified by        number of the following situations (none, one, or more than        one): age ≥65, presence of the following concerning medical        conditions: hypertension, chronic lung disease, obesity        [BMI≥30], diabetes, heart failure, coronary artery disease,        thrombotic events (current or by history), renal disease.        Patients will also be stratified by region in which they are        treated (US vs non-US). They will then be randomized 3:2 to        active drug or placebo at the schedule selected based on part A.        A total of approximately 250 additional patients will be        enrolled in part B of the study, 150 receiving active drug and        100 receiving placebo. Thus, combining both parts of the study,        a total of 170 patients will receive active at the dose selected        in part A and 120 will receive placebo. However, analyses will        be performed independently for parts A and B.        Patients will complete daily questionnaires about symptoms,        including adverse events, vital signs, including temperature and        pulse oximetry, and a log of medications taken, daily for the        first 4 weeks of study and thrice weekly thereafter. Viral swabs        and bloods for safety laboratory and pharmacodynamic markers        will be obtained at home visits by medical personnel. After        completion of treatment, patients will be followed through day        57 from randomization.        Statistics:        In part A of this study, two dose levels of active drug and        placebo will be tested. Based on the incidence and severity of        toxicities in each active group, overall assessment of safety by        the DSMB, a regimen for part B of the study will be selected. In        the absence of marked differences in toxicity between the two        active groups, the default choice for continuation into part B        will be the 400 mg daily regimen.        Efficacy data from parts A and B will be analyzed separately.        The overall sample size may be expanded based on interim study        results.        The sample size was determined based on the primary endpoint,        time to sustained recovery from COVID-19 illness, as defined in        the primary objective. It was calculated that in order to detect        an hazard ratio=1.5 comparing an active group to placebo group        with 3:2 allocation ratio a total of 201 recovery events are        required, to provide 80% power using a log-rank test at a        two-sided significance level of 0.05. Assuming 80% sustained        recovery rate by end of follow-up (assumed equal follow-up for        all enrolled patients), the minimum number of patients enrolled        in part B will be 250 in total, 150 in the active arm on the        regimen taken into part B of the study and 100 in the placebo        arm.

INDUSTRIAL APPLICABILITY

The present invention provides an anti-coronavirus agent comprising asan active ingredient a compound represented by:

as (L)- or (D)-enantiomers, and as E- or (Z)-isomers or (E/Z)-mixtures,and as free bases or as salts thereof, an anti-SARS agent comprising theanti-coronavirus agent and a method of treating SARS using theanti-coronavirus agent. The present invention enables the treatment ofdiseases caused by coronaviruses, especially the SARS-associatedcoronavirus.

All patents, patent applications, and published references cited hereinare hereby incorporated by reference in their entirety. Variousmodifications and variations of the described compositions and methodsof the invention will be apparent to those skilled in the art withoutdeparting from the scope and spirit of the invention. Although theinvention has been described in connection with specific embodiments, itwill be understood that the invention should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention that are obvious to thoseskilled in the fields of molecular biology, medicine, immunology,pharmacology, virology, or related fields are intended to be within thescope of the invention.

What is claimed is:
 1. A method for the treatment of the 2019coronavirus disease (COVID-19) caused by the SARS-CoV-2 virus comprisingadministering to a person in need thereof an effective amount of WX-671,

as (L)- or (D)-enantiomers, and as E- or (Z)-isomers or (E/Z)-mixtures,and as free bases or as salts thereof.
 2. The method of claim 1, whereinthe WX-671 is in the form of its hydrogen sulfate salt.
 3. The method ofclaim 1, further comprising a pharmaceutically-acceptable carriermaterial, wherein the WX-671 and the pharmaceutically-acceptable carriermaterial are in a unit dosage form suitable for oral administration. 4.The method of claim 2, further comprising a pharmaceutically-acceptablecarrier material, wherein the WX-671 hydrogen sulfate and thepharmaceutically-acceptable carrier material are in a unit dosage formsuitable for oral administration.
 5. The method of claim 3, wherein theunit dosage form is a solid dosage form.
 6. The method of claim 4,wherein the unit dosage form is a solid dosage form.
 7. The method ofclaim 5, wherein the solid dosage form is a capsule.
 8. The method ofclaim 6, wherein the solid dosage form is a capsule.
 9. The method ofclaim 1, wherein the SARS-CoV-2 virus is wild-type.
 10. The method ofclaim 1, wherein the SARS-CoV-2 virus is a naturally occurringcoronavirus variant.
 11. The method of claim 2, wherein the SARS-CoV-2virus is wild-type.
 12. The method of claim 2, wherein the SARS-CoV-2virus is a naturally occurring coronavirus variant.
 13. The method ofclaim 3, wherein the unit dosage form suitable for oral administrationis a capsule having 200 mg of WX-671 free base, and whereinadministering includes a single capsule administered once each day, forat least 10 days, for a total daily dose of 200 mg of WX-671.
 14. Themethod of claim 3, wherein the unit dosage form suitable for oraladministration is a capsule having 200 mg of WX-671 free base, andwherein administering includes two capsules administered once each day,for at least 10 days, for a total daily dose of 400 mg of WX-671. 15.The method of claim 4, wherein the unit dosage form suitable for oraladministration is a capsule having about 231 mg of WX-671 hydrogensulfate, and wherein administering includes a single capsuleadministered once each day, for at least 10 days, for a total daily doseof about 231 mg of WX-671 hydrogen sulfate.
 16. The method of claim 4,wherein the unit dosage form suitable for oral administration is acapsule having about 231 mg of WX-671 hydrogen sulfate, and whereinadministering includes two capsules administered once each day, for atleast 10 days, for a total daily dose of about 463 mg of WX-671 hydrogensulfate.
 17. A method of treatment comprising administering an effectiveamount of WX-671,

as (L)- or (D)-enantiomers, and as E- or (Z)-isomers or (E/Z)-mixtures,and as free bases or as salts thereof, to treat a human having 2019coronavirus disease (COVID-19) caused by the SARS-CoV-2 virus.
 18. Themethod of claim 17, wherein the WX-671 is in the form of its hydrogensulfate salt.
 19. The method of claim 17, further comprising apharmaceutically-acceptable carrier material, wherein the WX-671 and thepharmaceutically-acceptable carrier material are in a unit dosage unitform suitable for oral administration.
 20. The method of claim 18,further comprising a pharmaceutically-acceptable carrier material,wherein the WX-671 hydrogen sulfate and the pharmaceutically-acceptablecarrier material are in a unit dosage form suitable for oraladministration.
 21. The method of claim 19, wherein the unit dosage formis a solid dosage form.
 22. The method of claim 20, wherein the unitdosage form is a solid dosage form.
 23. The method of claim 21, whereinthe solid dosage form is a capsule.
 24. The method of claim 22, whereinthe solid dosage form is a capsule.
 25. The method of claim 17, whereinthe SARS-CoV-2 virus is wild-type.
 26. The method of claim 18, whereinthe SARS-CoV-2 virus is wild-type.
 27. The method of claim 17, whereinthe SARS-CoV-2 virus is a naturally occurring coronavirus variant. 28.The method of claim 18, wherein the SARS-CoV-2 virus is a naturallyoccurring coronavirus variant.
 29. The method of claim 20, wherein theunit dosage form suitable for oral administration is a capsule having anequivalent of 200 mg of WX-671, and wherein administering includes asingle capsule administered once each day, for at least 10 days, for atotal daily dose of about 400 mg of WX-671.
 30. The method of claim 20,wherein the unit dosage form suitable for oral administration is acapsule having an equivalent of 200 mg of WX-671, and whereinadministering includes two capsules administered once each day, for atleast 10 days, for a total daily dose of about 400 mg of WX-671.